The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA

Sloof, Paul; Haan, Annett de; Eier, Wilma; Iersel, Martijn P. van; Boel, Esper; Steeg, Harry van; Benne, Rob

doi:10.1016/0166-6851(92)90178-m

Cited by 43 publications

(24 citation statements)

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“…Thus, replication and assembly of the two types of these topologically linked kDNA circles requires a strict coordination between their replication mechanisms. Recently, two copies of an 11-mer sequence identical to UMS (apart from its 3Ј-terminal residue) were found in the maxicircle variable region of Trypanosoma brucei (46,47). The presence of this origin-associated sequence in both minicircles and maxicircles may provide a clue for understanding this coordination at the replication initiation step.…”

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Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids

Tzfati

Abeliovich

Avrahami

et al. 1995

Journal of Biological Chemistry

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Replication of kinetoplast DNA minicircles of trypanosomatids initiates at a conserved 12-nucleotide sequence, termed the universal minicircle sequence (UMS, 5-GGGGTTGGTGTA-3). A single-stranded nucleic acid binding protein that binds specifically to this origin-associated sequence was purified to apparent homogeneity from Crithidia fasciculata cell extracts. This UMS-binding protein (UMSBP) is a dimer of 27.4 kDa with a 13.7-kDa protomer. UMSBP binds single-stranded DNA as well as single-stranded RNA but not doublestranded or four-stranded DNA structures. Stoichiometry analysis indicates the binding of UMSBP as a protein dimer to the UMS site. The five CCHC-type zinc finger motifs of UMSBP, predicted from its cDNA sequence, are similar to the CCHC motifs found in retroviral Gag polyproteins. The remarkable conservation of this motif in a family of proteins found in eukaryotic organisms from yeast and protozoa to mammals is discussed.Kinetoplast DNA (kDNA) 1 is a unique extrachromosomal DNA network found in the single mitochondrion of parasitic flagellated protozoa of the family Trypanosomatidae. In Crithidia fasciculata, kDNA consists of about 5,000 DNA minicircles (2.5 kilobase pairs each) and about 50 DNA maxicircles (37 kilobase pairs each) interlocked topologically to form a huge DNA network (for review, see Refs. 1-3). Minicircles are heterogeneous in their nucleotide sequence but contain two short sequences, 70 -100 base pairs apart, that are conserved in all the species studied so far: the dodecamer sequence known as universal minicircle sequence (UMS), 5Ј-GGGGTTGGTGTA-3Ј, and the hexamer sequence 5Ј-ACGCCC-3Ј.On the basis of in vivo observations, Englund and co-workers (4 -8) have described the replication of kDNA minicircles as a process in which individual minicircles are detached from the central zone of the disc-shaped network, replicated, and reattached to the periphery of the disc. The network increases in size until it doubles and then divides and segregates into two daughter networks. Extensive studies of minicircle replication intermediates (9 -16) have suggested that replication begins at the UMS site with synthesis of an RNA primer and proceeds by continuous elongation of the leading light strand (L-strand). A single gap of 6 -10 nucleotides remains in the newly synthesized light strand at the UMS site (13) and is repaired only after replication of the minicircles and their reattachment to the network have been completed (8). Discontinuous synthesis of the lagging heavy strand (H-strand) starts when its origin, containing the conserved hexamer sequence, is exposed by the advancing replication fork. Highly gapped and nicked nascent H-strands are generated.We have previously reported on the recognition of UMS by a unique sequence-specific single-stranded DNA binding protein from C. fasciculata (17) and on the isolation and analysis of the UMSBP-encoding cDNA (18). The amino acid sequence of the polypeptide, predicted from the cDNA, is 116 residues long and contains five Cys-X 2 -Cys-X 4 -His-...

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Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids

Tzfati

Abeliovich

Avrahami

et al. 1995

Journal of Biological Chemistry

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“…The variable region is characterized by highly repetitive sequences (25, 37) and represents the major source of size and sequence divergence between species and within different stocks of the same species (2, 23). The entire sequence of the T. brucei maxicircle is known (11,25,35,37), whereas only about 20% of the C. fasciculata maxicircle has been sequenced (38,39,41).kDNA replication has been studied extensively (reviewed in references 29, 30, 32, and 33). In this process, covalently closed minicircles are released from the network by a topoisomerase II, and the free minicircles are thought to replicate within either of two complexes of replication enzymes which are situated on opposite sides of the network in vivo.…”

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Kinetoplast Maxicircle DNA Replication in Crithidia fasciculata and Trypanosoma brucei

Carpenter

Englund

1995

Molecular and Cellular Biology

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Kinetoplast DNA, the mitochondrial DNA of trypanosomatids, is composed of several thousand minicircles and a few dozen maxicircles, all of which are topologically interlocked in a giant network. We have studied the replication of maxicircle DNA, using electron microscopy to analyze replication intermediates from both Crithidia fasciculata and Trypanosoma brucei. Replication intermediates were stabilized against branch migration by introducing DNA interstrand cross-links in vivo with 4,5,8-trimethylpsoralen and UV radiation. Electron microscopy of individual maxicircles resulting from a topoisomerase II decatenation of kinetoplast DNA networks revealed intact maxicircle structures. Analysis of maxicircle DNA linearized by restriction enzyme cleavage revealed branched replication intermediates derived from structures. Measurements of the linearized branched molecules in both parasites indicate that replication initiates in the variable region (a noncoding segment characterized by repetitive sequences) and proceeds unidirectionally, clockwise on the standard map.The mitochondrial DNA of trypanosomes and related parasitic protozoa, known as kinetoplast DNA (kDNA), is organized into a single, massive network of catenated DNA circles (for reviews see references 29, 32-34, and 40). The network consists of several thousand minicircles (2.5 kb in Crithidia fasciculata; 1.0 kb in Trypanosoma brucei) and a few dozen maxicircles (38 kb in C. fasciculata; 23 kb in T. brucei). Maxicircles are similar in genetic function to the mitochondrial DNAs in higher eukaryotes. However, many maxicircle transcripts undergo editing, a unique process by which uridine residues are inserted at or deleted from specific sites to create functional open reading frames. Minicircles encode small guide RNAs that direct the editing of maxicircle transcripts (for reviews on editing see references 1, 13, 36, and 40).Maxicircles can be divided into two distinct regions. The coding region is 15 to 17 kb and contains the 12 and 9S rRNA genes as well as several protein coding genes. Most of the gene products, such as those for apocytochrome b and subunits of cytochrome oxidase, are involved in mitochondrial energy transduction. In contrast to mammalian mitochondrial DNA, there is no evidence for any tRNA genes within the maxicircle. The variable region is characterized by highly repetitive sequences (25, 37) and represents the major source of size and sequence divergence between species and within different stocks of the same species (2, 23). The entire sequence of the T. brucei maxicircle is known (11,25,35,37), whereas only about 20% of the C. fasciculata maxicircle has been sequenced (38,39,41).kDNA replication has been studied extensively (reviewed in references 29, 30, 32, and 33). In this process, covalently closed minicircles are released from the network by a topoisomerase II, and the free minicircles are thought to replicate within either of two complexes of replication enzymes which are situated on opposite sides of the network in vivo. Replicat...

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“…The mitochondrial genomes of many protozoal species, including leishmania, trypanosomes, and plasmodium, are unusual for their apparently total lack of tRNA genes (1)(2)(3). To sustain the translation of organellar mRNAs, a large number of nuclear-encoded tRNAs are imported from the cytoplasm (4 -10).…”

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Stepwise Transfer of tRNA through the Double Membrane of Leishmania Mitochondria

Mukherjee¹,

Bhattacharyya²,

Adhya

1999

Journal of Biological Chemistry

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Import of tRNA into Leishmania mitochondria involves transfer through a double membrane barrier. To examine whether specific sorting mechanisms for individual tRNAs direct them to different mitochondrial compartments, the distribution of tRNA transcripts, internalized in vitro, was examined by suborganellar fractionation. Significant amounts of tRNA Tyr were localized in the matrix and on the outer face of the inner mitochondrial membrane. With time, the matrix:membrane ratio increased. Translocation through the inner membrane apparently required the presence of a specific signal in the D arm of tRNA Tyr , and tRNA Gln (CUG), lacking this sequence, was excluded. Hydrolysis of ATP was necessary at both the outer and inner membranes. However, the protonophores carbonylcyanide m-chlorophenylhydrazone and nigericin, the K ؉ ionophore valinomycin, and the F 1 F 0 ATPase inhibitor oligomycin had only marginal effects on uptake through the outer membrane but severely inhibited inner membrane translocation, indicating the unusual requirement of both the electrical and chemical components of the electromotive force generated across the inner membrane. The results are consistent with a mechanism involving stepwise transfer of tRNA through distinct outer and inner membrane channels.

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The nucleotide sequence of the variable region in Trypanosoma brucei completes the sequence analysis of the maxicircle component of mitochondrial kinetoplast DNA

Cited by 43 publications

References 50 publications

Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids

Universal Minicircle Sequence Binding Protein, a CCHC-type Zinc Finger Protein That Binds the Universal Minicircle Sequence of Trypanosomatids

Kinetoplast Maxicircle DNA Replication in Crithidia fasciculata and Trypanosoma brucei

Stepwise Transfer of tRNA through the Double Membrane of Leishmania Mitochondria

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